The Stevens' laboratory has previously determined the three-dimensional structures of several forms of BoNT: (a) the 19S (900 kDa) serotype A toxin complex (therapeutic form) (11, 13), (b) the 150 kDa serotype A di- chain three domain neurotoxin (45), (c) the separate 50 kDa serotype B catalytic domain responsible for cleaving synaptic vesicle proteins (30), (d) the BoNT serotype B catalytic domain bound to its synaptic vesicle protein target (30), and (e) HA33 of serotype A. In addition, researchers from other laboratories have also determined the structures of other serotype proteins that are discussed below. Protocols used in protein preparation and crystallization for these studies will facilitate most of the work outlined below. In addition, these structures will be used as starting models for structure solution using the molecular replacement method. Recently, we have successfully cloned, expressed, purified and crystallized the light chains for BoNT Al (Figure 4). Thus we will be in a position to immediately start on determining its structure. A significant finding in the experiments leading to the crystallization of the domain was the requirement for the addition of Figure 4. Crystals of BoNT reducing agents to the buffer when concentrating as this avoided protein A1 (HaU> B8ht chain aggregation problems which had previously frustrated crystallization efforts. Ab-Toxin Interactions. In addition to these diffraction studies, we have previously performed Ab mapping studies on BoNT A that led to information on the domain organization of the toxin and how the holotoxin interacts with the neurotoxin associated proteins (NAPs) in the 19S (900 kDa) BoNT progenitor toxin complex (14). Forty-four monoclonal single-chain variable domain Ab fragments that bind to specific regions on each domain of the holotoxin were used to determine which domain regions were involved in packing interactions with other domains and with the NAPs in the BoNT progenitor toxin complex. Fifteen Ab binding sites were identified on the separated BoNT A catalytic domain, five Ab binding sites were identified on the translocation domain, and twenty-six Ab binding sites were identified on the binding domain. The Stevens laboratory has also had extensive experience working with Abs having determined the structures of a number of Ab-ligand structures as part of a long-term collaboration with Professor Schultz and Lerner in the area of catalytic Abs. The Stevens laboratory is therefore experienced in protocols for preparing Ab samples for crystallization studies. In this current project, attempts will be made to grow crystals of Abs complexed to BoNT (full length and domains). High-Throughput Structure Determination. Since the late 1990's, the Stevens laboratory has been involved in the development and deployment of new technology and approaches to X-ray crystallographic structure determination. Most notable was the development of high-throughput protein crystallization using submicroliter volumes of proteins (66, 59). Implemented on robotic systems capable of performing tens of thousands of experiments a day, this new approach has allowed the Joint Center for Structural Genomics (JCSG), where Dr. Stevens is co-Pi, to explore the whole proteome ofThermatoga maritima, solving the structure of over 150 of its proteins within a year. In addition to this crystallization work, the laboratory has been involved in JCSG's development of a highly parallel system capable of working with a large number of samples from the cloning and expression of proteins to the final refinement of a solved structure (46). We will use JCSG's approach and facilities to solve a large number of molecular species of BoNT as well as to conduct large-scale ligand-binding studies using potential inhibitors designed by the Kolb laboratory (Project 4).